IDENTIFICATION OF A LOCUS INVOLVED IN MENINGOCOCCAL LIPOPOLYSACCHARIDE BIOSYNTHESIS BY DELETION MUTAGENESIS

A novel method for insertion/deletion mutagenesis in meningococci was devised, This consisted of ligating a digest of total chromosomal DNA to a 1.1 kb restriction fragment containing an erythromycin-resistance marker (ermC), and subsequent transformation of the ligation mixture into the homologous meningococcal strain H44/76, Southern blotting of a number of the resulting erythromycin-resistant transformants demonst rated that all carried the ermC gene inserted at different positions i n the chromosome, Mutants with a specific phenotype were identified by screening with the anti-lipopolysaccharide (LPS) monoclonal antibody MN4A8B2, which is specific for immunotype L3. In this way, two indepen dent L3-negative mutant strains were isolated. In transformation exper iments with chromosomal DNA from these mutants, erythromycin-resistanc e and lack of MN4A8B2 reactivity were always linked, showing that the insertion/deletion was in a locus involved in LPS biosynthesis, On SDS -PAGE, the mutant LPS displayed an electrophoretic mobility intermedia te between that produced by the previously isolated galE and rfaF muta nt strains, Chemical analysis of the mutant LPS revealed that the stru cture was probably lipid A-(KDO)(2)-(Hep)(2). Chromosomal DNA flanking the ermC insertion in these two mutant strains was cloned, and used a s probe for the isolation of the corresponding region of the wild-type strain. From hybridization and polymerase chain reaction (PCR) analys is, it could be concluded that both mutations map to the same locus. T he affected gene probably encodes the glycosyltransferase necessary fo r adding N-acetylglucosamine to heptose.